Animal Endo-SiRNAs: Methods and Protocols by Andreas Werner

By Andreas Werner

Animal Endo-SiRNAs: tools and Protocols offers a number of techniques to enquire endo-siRNAs. those comprise protocols appropriate to review brief RNAs expressed at a low point and version platforms which are relatively compatible to enquire particular facets of endo-siRNAs, their synthesis, their genomics or regulatory function. Written within the hugely winning Methods in Molecular Biology series structure, chapters contain introductions to their respective subject matters, lists of the required fabrics and reagents, step by step, conveniently reproducible laboratory protocols and tips about troubleshooting and keeping off identified pitfalls.

Authoritative and useful, Animal Endo-SiRNAs: equipment and Protocols includes sensible counsel which are absent in common lab manuals.

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The increased depth of parallel sequencing developed in recent years, however, revealed that significant levels of endo-siRNAs can be detected—at least in selected tissues [4–6]. The biological phenomena associated with the formation of double-stranded RNA, transposon mobilization, and the expression of natural antisense transcripts show development and tissue specific expression. ), Animal Endo-siRNAs: Methods and Protocols, Methods in Molecular Biology, vol. 1007/978-1-4939-0931-5_3, © Springer Science+Business Media New York 2014 27 28 Sammer Alnumeir and Andreas Werner Fig.

Glycogen solution: 20 μg/μL (Roche Molecular Diagnostics). Microcentrifuge-based gel filtration columns : Removal of salts and other small molecules can be done efficiently using small gel filtration columns and a microcentrifuge. , Roche Mini Quick-Spin Oligo) as the columns must be RNase free. 2 Oxidation of Vicinal HydroxylGroups 1. 75 μL of NaIO4 solution (200 mM) (see Note 1) Incubate the mixture for 30 min at room temperature. Then add 3 μL of 100 % glycerol to quench the remaining NaIO4 and incubate for 10 min at room temperature.

Messenger RNA targets for these endo-siRNAs can be predicted with algorithms similar to those used for miRNA target predictions. Accordingly, endo-siRNA-directed target cleavage has been demonstrated but mRNA destabilization and/or translational inhibition may also be invoked. Thus, the biogenesis of these endosiRNAs resembles siRNAs whereas their mode of action is more akin to miRNAs. On the other hand, endo-siRNAs from complementary transcripts represent a complex mixture of small RNAs where each individual sequence is of low abundance.

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